Cohen Danielle, Hondelink Liesbeth M, Solleveld-Westerink Nienke, Uljee Sandra M, Ruano Dina, Cleton-Jansen Anne-Marie, von der Thüsen Jan H, Ramai S Rajen S, Postmus Pieter E, Graadt van Roggen Jacob F, Hoppe Bart P C, Clahsen Pieter C, Maas Klaartje W, Ahsmann Els J M, Ten Heuvel Alexandra, Smedts Frank, van Rossem Ronald N, van Wezel Tom
Department of Pathology, Leiden University Medical Centre (LUMC), Leiden, The Netherlands.
Department of Pathology, Leiden University Medical Centre (LUMC), Leiden, The Netherlands.
J Thorac Oncol. 2020 Jun;15(6):1000-1014. doi: 10.1016/j.jtho.2020.01.019. Epub 2020 Jan 31.
Frequently, patients with locally advanced or metastatic NSCLC are screened for mutations and fusions. In most laboratories, molecular workup includes a multitude of tests: immunohistochemistry (ALK, ROS1, and programmed death-ligand 1 testing), DNA sequencing, in situ hybridization for fusion, and amplification detection. With the fast-emerging new drugs targeting specific fusions and exon-skipping events, this procedure harbors a growing risk of tissue exhaustion.
In this study, we evaluated the benefit of anchored, multiplexed, polymerase chain reaction-based targeted RNA sequencing (RNA next-generation sequencing [NGS]) in the identification of gene fusions and exon-skipping events in patients, in which no pathogenic driver mutation was found by DNA-based targeted cancer hotspot NGS (DNA NGS). We analyzed a cohort of stage IV NSCLC cases from both in-house and referral hospitals, consisting 38.5% cytology samples and 61.5% microdissected histology samples, mostly core needle biopsies. We compared molecular findings in a parallel workup (DNA NGS and RNA NGS, cohort 1, n = 198) with a sequential workup (DNA NGS followed by RNA NGS in selected cases, cohort 2, n = 192). We hypothesized the sequential workup to be the more efficient procedure.
In both cohorts, a maximum of one oncogenic driver mutation was found per case. This is in concordance with large, whole-genome databases and suggests that it is safe to omit RNA NGS when a clear oncogenic driver is identified in DNA NGS. In addition, this reduced the number of necessary RNA NGS to only 53% of all cases. The tumors of never smokers, however, were enriched for fusions and exon-skipping events (32% versus 4% in former and current smokers, p = 0.00), and therefore benefited more often from the shorter median turnaround time of the parallel approach (15 d versus only 9 d in the parallel workup).
We conclude that sequentially combining DNA NGS and RNA NGS is the most efficient strategy for mutation and fusion detection in smoking-associated NSCLC, whereas for never smokers we recommend a parallel approach. This approach was shown to be feasible on small tissue samples including for cytology tests, can drastically reduce the complexity and cost of molecular workup, and also provides flexibility in the constantly evolving landscape of actionable targets in NSCLC.
局部晚期或转移性非小细胞肺癌（NSCLC）患者常接受突变和融合检测。在大多数实验室中，分子检测包括多项测试：免疫组织化学（ALK、ROS1和程序性死亡配体1检测）、DNA测序、融合原位杂交以及扩增检测。随着针对特定融合和外显子跳跃事件的新型药物迅速涌现，这一检测流程面临着组织耗尽风险不断增加的问题。
在本研究中，我们评估了基于锚定多重聚合酶链反应的靶向RNA测序（RNA下一代测序[NGS]）在鉴定DNA靶向癌症热点NGS（DNA NGS）未发现致病驱动突变的患者基因融合和外显子跳跃事件中的作用。我们分析了来自内部医院和转诊医院的一组IV期NSCLC病例，其中38.5%为细胞学样本，61.5%为显微切割组织学样本，大多为粗针活检样本。我们将平行检测（DNA NGS和RNA NGS，队列1，n = 198）与序贯检测（在选定病例中先进行DNA NGS，然后进行RNA NGS，队列2，n = 192）的分子检测结果进行了比较。我们假设序贯检测是更有效的方法。
在两个队列中，每个病例最多发现一种致癌驱动突变。这与大型全基因组数据库一致，表明当在DNA NGS中鉴定出明确的致癌驱动因素时，省略RNA NGS是安全的。此外，这将必要的RNA NGS检测数量减少至所有病例的仅53%。然而，从不吸烟者的肿瘤中融合和外显子跳跃事件更为丰富（从不吸烟者中为32%，既往吸烟者和当前吸烟者中为4%，p = 0.00），因此更常受益于平行检测方法较短的中位周转时间（平行检测中为15天，序贯检测中仅为9天）。
我们得出结论，对于吸烟相关的NSCLC，序贯联合DNA NGS和RNA NGS是检测突变和融合的最有效策略，而对于从不吸烟者，我们建议采用平行检测方法。该方法在包括细胞学检测在内的小组织样本上被证明是可行的，可大幅降低分子检测的复杂性和成本，并且在NSCLC中可操作靶点不断变化的情况下也提供了灵活性。
